In order to fully understand the mechanism of in vitro motility in the sliding actin assay and to ascertain to what extent the assay mimics the behavior of intact muscle, it is necessary to compare the behavior of both systems. To accomplish this, we have characterized the behavior of rabbit skeletal muscle HMM in the in vitro motility assay and compared its movement with the unloaded shortening velocity of skeletal muscle fibers. We find that, in most cases, the in vitro assay mimics the unloaded shortening-velocity, but there are several specific cases where the two systems behave distinctly different. We examined the movement of HMM as a function of ionic strength, pH, temperature and MgATP concentration. The movement of actin by HMM was sensitive to changes in each of these conditions, but, in most cases, the velocity of the in vitro motility assay paralleled that of the unloaded shortening velocity of vertebrate muscle fibers under similar conditions. The major exceptions were at low pH (below 6.5) where in vitro motility ceased, but where muscle fibers still shorten and, at lower temperatures, where the activation energy is greater for the motility assay than for muscle fibers. At present, we have no explanation for these differences.